Non-receptor tyrosine kinases (NRTKs) function as tightly regulated, integrating switches in cellular signal transduction. Here we focus on representatives of three NRTK families, which have distinct multi-domain architectures: Jak-family kinases, Focal Adhesion Kinase (FAK), and the Tec-family member ITK (Il-2 inducible tyrosine kinase). Our longterm goals are to elucidate autoregulatory mechanisms of NRTKs at a structural level, to understand how these regulatory mechanisms are disrupted in cancer, and to use structural insights to facilitate discovery of novel inhibitors. With our collaborators, we are combining the tools of structural biology, biochemistry, cell biology, and chemistry in a unified way to advance these goals. We propose three Specific Aims. First, we will discover how Jak kinases recognize their cognate cytokine receptors and how they are regulated by interactions among their constituent domains. We have crystallized an Nterminal fragment of Jak2, and have also prepared a complex of this portion of Jak2 with the cytoplasmic tail of the erythropoietin receptor for structural analysis. Our studies of Jak regulation build on our recently determined structure of a linker/pseudokinase regulatory module, and through elucidation of the structure of essentially full-length Jak2, we will explain how the this module controls the activity of the adjacent kinase domain, and how this control is disrupted by the V617F mutation in myeloproliferative neoplasms. Second, we will determine structurally how focal adhesion kinase (FAK) is activated by PI(4,5)P2. This aim builds on our determination of the structure of FAK in its autoinhibited state, and our discovery that it binds and is activated by the PI(4,5)P2. Third, we will elucidate the structure an SH3-SH2-kinase fragment of Itk in order to understand its regulation and to facilitate inhibitor discovery. Based on our structural insights, we are developing irreversible inhibitors specific for Jak3 that may be useful in treatment of autoimmune and inflammatory disorders.